1. Field of the Invention
This invention relates to and has among its objects the provision of a novel method for separating alpha-1-proteinase inhibitor (PI) from blood plasma or blood plasma fractions. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
Alpha-1-proteinase inhibitor is a glycoprotein having molecular weight of 54,000. The protein consists of a single polypeptide chain to which several oligosaccharide units are covalently bound. Human PI has a role in controlling tissue destruction by endogenous serine proteinases. A genetic deficiency of PI, which accounts for 90% of the trypsin inhibitory capacity in blood plasma, has been shown to be associated with premature development of pulmonary emphysema. The degradation of elastin associated with emphysema probably results from a local imbalance of elastolytic enzymes and the naturally occurring tissue and plasma proteinase inhibitors. PI rapidly inhibits human pancreatic and leukocyte elastases (Biochem. Biophys. Res. Comm., Vol. 72, No. 1, pages 33-39, 1976; ibid., Vol. 88, No. 2, pages 346-350, 1979).
A number of methods have been employed to isolate PI from the blood plasma. A majority of these methods are directed to laboratory scale isolation while others pertain to production on a commercial level.
Pannell et al., Biochemistry, Vol. 13, pages 5439-5445, (1974), employed a process wherein albumin-poor blood plasma was pooled and fractionated with solid ammonium sulfate (0.60-0.80 saturation). The precipitate resulting was solubilized and dialyzed and applied to a column of DEAE-cellulose. The 0.05-0.15 M NaCl linear gradient is pooled, concentrated, and dialyzed, and then applied again to a column of DEAE-cellulose. The linear gradient from 0.05-0.20 NaCl was collected, pooled, and concentrated to give PI.
In the method of Saklatvala et al., Biochem. J., Vol. 157, pages 339-351 (1976), human plasma was fractionated using ammonium sulfate (80% saturation) to give a precipitate, which was dissolved, dialyzed and chromatographed on DEAE-cellulose. The 0.5 M NaCl extract was applied to a concanavalin A-Sepharose column. The alpha-D-methyl glucopyranoside eluate was concentrated and applied again to a DEAE-cellulose column. The 0.0-0.2 M NaCl eluate contained PI.
Fifty percent saturated ammonium sulfate precipitation was used by Musiani et al., Biochem., Vol. 15, pages 798-804 (1976) to separate a PI-rich fraction that was solubilized and then subjected to successive chromatographic steps using DEAE ion exchanger, concanavalin A-Sepharose, Sephadex G-100, and an immunoadsorbent column to yield purified PI.
A large scale purification of PI from human plasma was disclosed by Kress et al., Preparative Biochemistry, Vol. 3, No. 6, pages 541-552 (1973). The precipitate from the 80% ammonium sulfate treatment of human plasma was dialyzed and chromatographed on DEAE-cellulose. The concentrate obtained was again dialyzed and gel filtered on Sephadex G-100. The PI-containing fractions were chromatographed twice on DE-52 cellulose to give PI.
Glaser et al., ibid., Vol. 5, No. 4, pages 333-348 (1975) isolated PI from Cohn Fraction IV-1 in 30% overall yield. Dissolved IV-1 was chromatographed on DEAE-cellulose, QAE-Sephadex, concanavalin A-Sepharose, and G-150 Sephadex to give PI.
An integrated plasma fractionation system based on polyethylene glycol (PEG) was disclosed by Hao et al., Proceedings of the International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation, held Sept. 7-9, 1977, Reston, Va. In the published method Cohn cryoprecipitate was mixed with PEG in an amount of 40 grams per liter (g/l). All operations were conducted at 5.degree. C.
After stirring for 60 minutes, the first fraction was removed by centrifugation. An additional 60 g/l of PEG was added to the supernate (final concentration approximately 10%). Prothrombin complex (PTC) was then extracted from the 10% PEG supernate by batch-wise adsorption on DEAE cellulose, and an additional 100 g/l of PEG was added to obtain the 10-20% PEG precipitate. The four fractions thus obtained were 0-4% PEG precipitate, 4-10% PEG precipitate, 10-20% PEG precipitate and 20% PEG supernate, and were designated as Fractions A, B, C and D, respectively. It should be pointed out that these PEG concentrations were based on the original volume of cryosupernate.
The distribution of proteins in the four PEG fractions was as follows: Fibrinogen was the dominant protein in Fraction A with albumin being the major contaminant. Most of the contaminating albumin in Fractions A, B and C resulted from coprecipitation and/or entrapment of supernate since albumin by itself did not precipitate under these conditions. Fraction B was rich in plasminogen, C3 component of complement, IgG and IgM. In addition, virtually all of the beta-lipoproteins were present in this fraction. Fraction C contained appreciable quantities of alpha.sub.2 macroglobulin, IgA and was rich in prothrombin and other coagulation factors which constitute the so-called prothrombin complex. However, the authors found that better yields of PTC could be obtained from the 10% PEG supernate rather than from the 10-20% PEG precipitate. Fraction D was dominated by albumin but also contained all of the alpha-1-acid glycoprotein as well as most of the PI, antithrombin III (AT III), ceruloplasmin (C.sub.p), haptoglobin, transferrin (T.sub.f) and Cl esterase inhibitor (Cl inhib.). Several additional proteins were also isolated from Fraction D including prealbumin (PA), retinol binding protein (RBP), transcortin, and angiotensinogen. In general, most of the smaller proteins were in Fraction D.